EP0220657B1 - Reaktionsverbindung von 9-(2-Hydroxyethoxymethyl)-guanin mit lactosaminiertem menschlichem Albumin, Verfahren zu ihrer Herstellung und sie enthaltende pharmazeutische Präparate - Google Patents

Reaktionsverbindung von 9-(2-Hydroxyethoxymethyl)-guanin mit lactosaminiertem menschlichem Albumin, Verfahren zu ihrer Herstellung und sie enthaltende pharmazeutische Präparate Download PDF

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Publication number
EP0220657B1
EP0220657B1 EP86114557A EP86114557A EP0220657B1 EP 0220657 B1 EP0220657 B1 EP 0220657B1 EP 86114557 A EP86114557 A EP 86114557A EP 86114557 A EP86114557 A EP 86114557A EP 0220657 B1 EP0220657 B1 EP 0220657B1
Authority
EP
European Patent Office
Prior art keywords
conjugate
hydroxyethoxymethyl
guanine
human albumin
acv
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP86114557A
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English (en)
French (fr)
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EP0220657A3 (en
EP0220657A2 (de
Inventor
Massimo Baldacci
Luigi Fiume
Corrado Busi
Alessandro Mattioli
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Laboratori Baldacci SpA
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Laboratori Baldacci SpA
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Publication date
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Priority to AT86114557T priority Critical patent/ATE66152T1/de
Publication of EP0220657A2 publication Critical patent/EP0220657A2/de
Publication of EP0220657A3 publication Critical patent/EP0220657A3/en
Application granted granted Critical
Publication of EP0220657B1 publication Critical patent/EP0220657B1/de
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Expired - Lifetime legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]

Definitions

  • the present invention relates to a novel compound, namely the conjugation product of 9- (2-hydroxyethoxymethyl)-guanine with lactosaminated human albumin, useful in the therapeutical treatment of chronic hepatitis B.
  • 9-(2-hydroxyethoxymethyl)-guanine also known as acyclovir (ACV), having the formula: is an anti-viral drug of widespread use in the treatment of herpetic infections (Dolin L., Science 227, 1296-1303 (1985). Recently it has also been used in the treatment of chronic hepatitis B with good results (Weller I.V.D. et al., Lancet i, 273, (1982), and Weller I.V.D. et al, J. Antimicr. Chem. 11, 223-231, (1983)), mainly in association with interferon (Schalm S.W. et al, Lancet ii, 358-360, (1985)).
  • ACCV acyclovir
  • ACV at the dosages at which the reproduction of the virus of hepatitis B (HBV) is inhibited, (5-15 mg/kg), may cause renal lesions (Weller I.V.D. et al, J. Antimicr. Chem. 11, 223-231, (1983); Balfour H.H., Ann. Rev. Med. 35, 279-291, 1984) and alterations of the central nervous system (Balfour, ibidem.).
  • Lactosaminated human albumin is a neoglycoprotein which selectively penetrates the hepatocytes after interaction with a receptor specific for the proteins ending with galactose present only on the parenchimal cells of liver (Wilson G., J. Biol. Chem. 253, 2070, (1978)). It has been sucessfully used in the mouse as the hepatotropic vehicle (carrier) of adenine arabinoside monophosphate (ARA-AMP) (Fiume L. et al, FBS Lett. 129, 261-264 (1981); Fiume L. et al., Gut 25, 1392-1398, (1984)).
  • ARA-AMP adenine arabinoside monophosphate
  • the dose of conjugated acyclovir to be administered in order to inhibit the synthesis of viral DNA in the liver is about 6% of that of free acyclovir capable of achieving the same inhibition degree.
  • the conjugate according to the present invention seems to be capable of increasing the chemotherapeutical index of ACV in the treatment of chronic hepatitis B with a substantial reduction or elimination of the aforesaid side-effects, intolerable or highly disturbing for the patient.
  • an object of the present invention is the process for the preparation of the conjugate, comprising the use of a derivative of acyclovir, preferably selected from one of monophosphate succinate and glutarate, and the bonding of the derivative to the lactosaminated human albumin by means of carbodiimides or, as an alternative (in the case of succinate and glutarate), by means of their hydroxysuccinimidic ester, or by the mixed anhydride method.
  • a derivative of acyclovir preferably selected from one of monophosphate succinate and glutarate
  • the bonding of the derivative to the lactosaminated human albumin by means of carbodiimides or, as an alternative (in the case of succinate and glutarate), by means of their hydroxysuccinimidic ester, or by the mixed anhydride method.
  • the monosphosphate derivative of acyclovir is bonded to the lactosaminated albumin by means of 1-ethyl-3-(dimethylaminopropyl)-carbodiimide (ECDI), the reaction being carried out in aqueous solution at a pH higher than 6.5.
  • ECDI 1-ethyl-3-(dimethylaminopropyl)-carbodiimide
  • lactosaminated albumin and the formation of conjugates therefrom further details can be seen from e.g. EP-A-184838.
  • lactosaminated human albumin a human albumin is meant those in which at least 20, more preferably 30, of the 59 lysine residues existing in that albumin are linked to galactose molecules through the glucose moiety.
  • ACV monophosphate (ACV-MP) was obtained by adding phosphorous oxychloride to a cooled suspension (-2°C) of ACV in triethylphospate (Yoshikawa M. et al, Tetrahedon Letters 50, 5065-5068, (1967) and Le Page G.A. et al, Cancer Res. 35, 3036-3040, 1975)). It was purified by chromatography through a column of DOWEX®-1 formiate eluted with a continuous gradient of formic acid (O to 4M). Lactosaminated human albumin with 30 sugar residues (L30-HSA) was obtained by reductive amination (Schwartz B-A. et al.,Arch. Biol. Chem. Biophys. 181, 542-549, (1977)).
  • ACV-MP was conjugated with L30-HSA by the following process: 1 g (3.27 mmoles) of ACV-MP and 1 g (12.6 ⁇ moles) of L30-HSA were dissolved in 20 ml H2O. The pH was adjusted to 7.5 with 10N NaOH and 1 g (5.21 mmoles) ECDI was added. After 24 hours' incubation at 25°C under stirring and in the dark, the reaction mixture was dialyzed against 0.9% NaCl to remove the unbonded ACV-MP, the ECDI and its ureic derivative. When the dialysate no longer showed an absorption at 255 nm the conjugate was collected and concentrated to 500 mg/10ml in minicon B-15 (Amicon) cells. 1 ml fractions (containing 50 mg conjugate and 9 mg NaCl) are lyophilized. At the time of use, a fraction is dissolved in 1 ml H2O and, if necessary, further diluted with 0.9% NaCl.
  • the albumin concentration is measured by the method of Lowry et al (J. Biol. Chem. 193, 265-275,1951).
  • the molar ratio ACV/L30-HSA in several conjugate preparations has been found to vary between 10 and 12:1.
  • Fetuine was enzymatically desialated (Morrell A.G. et al, J. Biol. Chem. 241, 3745-3749 (1966)). Then it was marked with [14C] formaldehyde according to Cox. R.A. and Greenwell P., Biochem. J. 186, 861-872 (1980). Swiss female mice of 28-30 g were treated by the intravenous route with 2 ⁇ g/g of [14C] AF (4.9 x 106 dpm/mg).
  • L30-HSA and the conjugate were intravenously administered at the dose of 2 ⁇ g/g simultaneously with [14C] AF. In all cases the injected volume was 10 ⁇ l/g. After 10 minutes blood samples were taken from the retroorbital plexus of the animals under ether-induced anaesthesia and the radioactivity of the plasma was measured. Each value represents the average ( ⁇ standard error) of the results obtained in 5 animals. The difference between the results of animals treated with L30-HSA and those of the animals administered with the conjugate, as evaluated by the Student t values, is significant (P ⁇ 0.02). (For details of the Student t test see "Statistical Methods in Medical Research", 2nd Ed., Blackwell Scientific Publications, Oxford, 1987, p.98-100 and 186-192.)
  • Table 2 shows the effect of ACV, ACV-MP and of the conjugate L30-HSA -ACV-MP10 on the incorporation of thymidine in the DNA of liver, intestine and medulla ossium of mice affected by hepatitis induced by Ectromelia virus. In these animals the incorporation of thymidine in the liver is caused by the synthesis of viral DNA (Fiume L. et al, FEBS Lett. 129-261-264, XX (1981)).
  • the lowest dose of ACV causing a significant inhibition of the DNA synthesis in the liver was 10 ⁇ g/g, corresponding to that used in patients affected by chronic hepatitis B.
  • ACV also inhibits DNA synthesis in the intestine to a significant extent.
  • ACV-MP in free form was not more active than ACV.
  • ACV-MP conjugated with L30-HSA caused the inhibition of DNA synthesis in the liver at a dose of 0.6 ⁇ g/g only. Moreover no significant inhibition in the intestine and medulla ossium was induced.
  • Conjugates of ACV with lactosaminated albumin can also be obtained according to processes different from that described herein.
  • ACV may be converted, by means of glutaric anhydride, to ACV-glutarate, or by means of the succinic anhydride to the ACV-succinate.
  • these derivatives can be bonded to the L-SA either through their hydroxysuccinimidic ester, or through the mixed anhydride method or by using carbodiimides.
  • These processes have been already used for the bonding of ACV to bovine and rabbit albumins and to human immunoglobulins (Quinn, R.P. et al.,Analyt. Biochem. 98 , 319-328 (1979)).
  • the resulting derivatives have been used as immunogens to obtain antibodies retaining ACV which are used for the radioimmunological dosing of this drug.
  • adenine arabinoside (Ara-A)
  • Ara-A adenine arabinoside
  • a conjugate ara-A-glut-AF has been obtained which proved to be pharmacologically active in inducing hepatic targeting of ara-A in the mouse (Fiume et al., FEBS Lett. 116 , 185-188 (1980)).

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Claims (7)

  1. Reaktionsverbindung von 9-(2-Hydroxyethoxymethyl)-guanin mit lactosaminiertem menschlichem Albumin mittels einer Brücken-Einheit, ausgewählt aus Monophosphat, Succinat und Glutarat.
  2. Reaktionsverbindung gemäß Anspruch 1, dadurch gekennzeichnet, daß das molare Verhältnis von 9-(2-Hydroxyethoxymethyl)-guanin zum lactosaminierten Albumin zwischen 10:1 und 12:1 liegt.
  3. Verfahren zur Herstellung der Reaktionsverbindung gemäß einem oder beiden der Ansprüche 1 bis 2, dadurch gekennzeichnet, daß wäßrige Lösungen eines Derivates von 9-(2-Hydroxyethoxymethyl)-guanin und des lactosaminierten menschlichen Albumins unter Rühren und in der Dunkelheit umgesetzt werden.
  4. Verfahren gemäß Anspruch 3, dadurch gekennzeichnet, daß das Derivat von 9-(2-Hydroxyethoxymethyl)-guanin aus einer der Verbindungen mit Monophosphat, Glutarat und Succinat ausgewählt wird.
  5. Verfahren gemäß Anspruch 3, dadurch gekennzeichnet, daß das Derivat von 9-(2-Hydroxyethoxymethyl)-guanin das Monophosphat ist und die Reaktion mit lactosaminiertem menschlichem Albumin in Gegenwart von 1-Ethyl-3-(diethylaminopropyl)-carbodiimid 24 h bei einem pH über 6,5 bei 25°C in der Dunkelheit und unter Rühren durchgeführt wird.
  6. Pharmazeutische Zusammensetzung, dadurch gekennzeichnet, daß sie als aktiven Bestandteil die Reaktionsverbindung gemäß den Ansprüchen 1 oder 2 in Kombination mit üblichen Trägern und Vehikeln enthält.
  7. Pharmazeutische Zusammensetzung gemäß Anspruch 6, die bei der Therapie der chronischen Hepatitis B verwendbar ist.
EP86114557A 1985-10-21 1986-10-21 Reaktionsverbindung von 9-(2-Hydroxyethoxymethyl)-guanin mit lactosaminiertem menschlichem Albumin, Verfahren zu ihrer Herstellung und sie enthaltende pharmazeutische Präparate Expired - Lifetime EP0220657B1 (de)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AT86114557T ATE66152T1 (de) 1985-10-21 1986-10-21 Reaktionsverbindung von 9-(2-hydroxyethoxymethyl)-guanin mit lactosaminiertem menschlichem albumin, verfahren zu ihrer herstellung und sie enthaltende pharmazeutische praeparate.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT8522559A IT1207507B (it) 1985-10-21 1985-10-21 Coniugato della 9-(2-idrossietossimetil)guanina con albumina umana lattosaminata, procedimento per la sua preparazione e composizioni farmaceutiche che la contengono.
IT2255985 1985-10-21

Publications (3)

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EP0220657A2 EP0220657A2 (de) 1987-05-06
EP0220657A3 EP0220657A3 (en) 1988-10-05
EP0220657B1 true EP0220657B1 (de) 1991-08-14

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EP86114557A Expired - Lifetime EP0220657B1 (de) 1985-10-21 1986-10-21 Reaktionsverbindung von 9-(2-Hydroxyethoxymethyl)-guanin mit lactosaminiertem menschlichem Albumin, Verfahren zu ihrer Herstellung und sie enthaltende pharmazeutische Präparate

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US (1) US4725672A (de)
EP (1) EP0220657B1 (de)
AT (1) ATE66152T1 (de)
DE (1) DE3680857D1 (de)
ES (1) ES2037654T3 (de)
IT (1) IT1207507B (de)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT1245217B (it) * 1991-03-12 1994-09-13 Baldacci Lab Spa Coniugato di azidotimidina ed albumina umana procedimento per la sua preparazione e composizioni farmaceutiche che lo contengono
IT1256110B (it) * 1992-11-23 1995-11-28 Procedimento di coniugazione di nucleosidi antivirali con albumina umana lattosaminata
ITMI20010027A1 (it) * 2001-01-10 2002-07-10 Uni Di Bologna Dipartimento Di Composizioni teraputiche per una chemioterapia locoregionale non invasiva delle micrometastasi epatiche
US20140271772A1 (en) * 2013-03-15 2014-09-18 Barry J. Margulies Biodegradable subcutaneous implants and methods of making

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* Cited by examiner, † Cited by third party
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GB1523865A (en) * 1974-09-02 1978-09-06 Wellcome Found Purine compunds and salts thereof
IT1177381B (it) * 1984-12-11 1987-08-26 Baldacci Lab Spa Metodo per la preparazione di coniugati della adenina-9-beta-d-arabino furano-side 5' monofosfato con albumina umana lattosaminata, coniugati risultanti e relative composizioi terapeuticamente attive

Also Published As

Publication number Publication date
DE3680857D1 (de) 1991-09-19
US4725672A (en) 1988-02-16
ATE66152T1 (de) 1991-08-15
IT1207507B (it) 1989-05-25
EP0220657A3 (en) 1988-10-05
ES2037654T3 (es) 1993-07-01
IT8522559A0 (it) 1985-10-21
EP0220657A2 (de) 1987-05-06

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